|Molecular Vision 2001;
Received 4 February 1999 | Accepted 17 November 2001 | Published 20 November 2001
Altered expression of alternatively spliced isoforms of the mRNA NMDAR1 receptor in the visual cortex of strabismic cats
Zheng Qin Yin,1,2 Ze Min
Deng,3 Sheila G. Crewther,1,4
David P. Crewther1,4,5
Schools of 1Optometry and 3Pathology, University of New South Wales, Sydney, Australia; 2Department of Ophthalmology, Southwest Hospital, Chongqing, China; 4School of Psychological Science, La Trobe University, Melbourne, Australia; 5Brain Sciences Institute, Swinburne University of Technology, Melbourne, Australia
Correspondence to: Zheng Qin Yin, Department of Ophthalmology, Southwest Hospital, Gaotanyan Street, Chongqing 400038, China; Phone: 86-23-68754701; FAX: 86-23-68754701; email: email@example.com
Purpose: Although much has been written about the role of the NMDA receptor's role in experience dependent visual plasticity, the function of the NMDAR1 receptor subunit in the post-plasticity stage of development is still not well understood. However, in the well studied model of strabismic amblyopia where binocularity is reduced, but where most primary visual cortex neurons can be driven by one or other eye, the density of expression of NMDAR1 receptor protein is significantly reduced, compared to normals. This study aims to identify which of eight isoforms of the spliced heterogeneous variants of the NMDAR1 mRNA receptor gene are associated with this decrease in expression as a means of elucidating possible function.
Methods: A series of digoxygenin-labelled oligonucleotide probes based on the human gene sequence have been used for in situ hybridization (ISH) of sections from the striate cortex of four adult cats. The probes were used to uniquely detect the expression of alternatively spliced mRNA variants in 66,487 cells from sections from the area centralis projection of two normal cats and two cats made esotropic as kittens by tenotomy at two weeks of age.
Results: As expected, total NMDAR1 mRNA isoform expression was significantly lower in the striate cortex of strabismic compared to normal cats. The proportion of cortical cells expressing the R1-a, R1-b, and R1-1 isoforms in strabismic animals was decreased while the proportion expressing R1-3 was increased, especially in layers V and VI. No significant difference in expression of the R1-2 and R1-4 isoforms was seen comparing strabismic and normal cats.
Conclusions: These results confirm our previous findings and suggest that transcriptional inhibition of specific isoforms of NMDAR1 mRNA may underlie the change in receptor expression. This preferential reduction in the proportion of neurons bearing particular NMDAR1 isoforms, i.e. isoforms R1-a and b, and R1-1 with partial compensation through the expression of the R1-3 isoform, is more likely related to lowered proportion of binocularly activated neurons in the strabismic cat than to changes in eye dominance or the presence of amblyopia in one eye.
The N-methyl-D-aspartate (NMDA) glutamate receptors mediate excitatory neurotransmission in the brain and are thought to play an important role in neuronal development, normal neurotransmission, learning and memory . In particular, the activation of NMDA receptors has been proposed as a prerequisite for the induction of experience dependent modification of neuronal responses within the visual cortex during the critical period of early postnatal development , as blockade of the receptor reduces the degree of monocular occlusion induced shift in ocular dominance .
NMDA receptors are highly complex multimeric proteins with two major subunits, NMDAR1 and NMDAR2 . The NMDAR1 subunit confers the major characteristic properties of the NMDA receptor including Ca2+ permeability, while the NMDAR2 subunit confers functional variation including altered receptor kinetics over the critical period of visual development [4,5]. Catalano et al. have also described developmental changes in the laminar pattern of NMDAR1 immunoreactivity in cat neocortex with a gradual decline in immunostaining of cortical layers V and VI with age, transient increase in layer IV staining between P34-53 and maintenance of higher immunostaining levels in layers II-III through to adulthood. They also reported that while dark rearing of kittens did not cause changes in NMDAR1 immunoreactivity, two weeks of monocular inactivation of spiking neurons induced by tetrodotoxin administration caused a decrease in NMDAR1 immunoreactivity in striate cortical columns corresponding to the blocked eye . Brief periods of visual experience in dark reared rat pups results in changes in the NMDAR2A/R2B ratio with consequent changes in synaptic kinetics . Chen et al also found dark rearing to not alter the level of NMDAR1 and NMDAR2B subunits, but that NMDAR2A expression was elevated in normal compared with dark reared visual cortex at 5 weeks of age. This relation changed at 20 weeks, with the level of NMDAR2B in dark reared cortex twice that of normal  supporting the idea of involvement of this subunit in critical period plasticity in visual cortex.
The involvement of NMDAR1 in synaptic plasticity was also supported by further immunohistochemical studies showing a patchy distribution of NMDAR1 subunit in kittens at 2 weeks of age. This distribution pattern was found to be susceptible to the effects of monocular deprivation (by lid suture) . The high density patches tended to not correlate with the centers of the ocular dominance bands, but rather with the borders of columns leading Trepel and colleagues to suggest that the NMDAR1 patches may participate in sculpting the arrangement of visual cortical columns .
The situation in amblyopic cats is somewhat different. We reported a reduction in the total percentage of neurons positively labelled for the NMDAR1 antibody in adult amblyopic cats raised from 2 weeks of age with either strabismus (surgically induced) or anisometropia (lens induced) [9,10]. In the normal cat, the distribution of NMDAR1 immunopositivity was most prominent in layer II and upper layers III and IV, whereas in strabismic cats, NMDAR1 immunopositive cells were seen predominantly in layers II and III. In anisometropic cats, NMDAR1 immunopositive cells also appeared predominantly in layer II to layer III, with a few more positively staining cells in layers IV, V, and VI than were seen in the strabismic cat.
Recent in situ hybridization experiments have demonstrated eight splice variants of the mRNA gene protein associated with the NMDAR1 receptor in rat and human . It has been proposed that these eight isoforms have different functional properties in agonist affinity, current amplitude, modulation by Zn2+, potentiation by polyamines and regulation by protein kinase C (PKC) [12-14]. Thus we considered it important to utilize in situ hybridization (ISH) techniques to further identify which isoform(s) of NMDAR1 mRNA are associated with the decrease in the NMDAR1 receptor previously described in visual cortex of strabismic cat, as a means to elucidating the functional changes of NMDA receptors in amblyopia.
Over the last few years ISH techniques have been developed to identify the cellular source of the expression of specific mRNA species  in brain tissue. Originally, radioactively labelled isotopic probes were used, however a new non-isotopic probe technique has substantially improved the cellular localization of the label , prolonged storage life of the labelled probe to months, and reduced radioactive waste. Thus, the specific aim of this experiment was to use a non-isotopic ISH technique to analyze the relative contributions of the eight isoforms of NMDAR1 to the overall reduced density of NMDAR1 immunopositive labeling in the visual cortex of strabismics cats.
Two normal and two strabismic adult cats were used in this study. The strabismic cats were made monocularly esotropic (by tenotomy) [17,18] at two weeks of age. The kittens grew to adult weight of 1.5 to 2.0 kg by 6 months of age with a pronounced squint measuring about 15° to 20°, resulting in physiological amblyopia confirmed by Multifocal VEP (VERIS, Electro-Diagnostic Imaging, San Mateo, USA). Animals were sacrificed with an overdose of Nembutal (100 mg/kg) given intraperitoneally. Animal rearing and experimental conduct were in accordance with the Australian NH & MRC guidelines and the ARVO convention on animal use.
Fresh, frozen sections (20 mm thick) from regions of primary visual cortex, P5-P0, corresponding to the site of the area centralis projection were cut on a cryostat at -15 °C. Sections were mounted on slides that were then washed in acetone and air dried at room temperature for 5 min before immersion in a coating solution (2% 3-amino-propyltriethoxysilane) for 5 min, followed by 10 dips in acetone and 10 dips in diethylpyrocarborate (DEPC) treated water. The sections were again air dried, then fixed in freshly prepared 4% paraformaldehyde in 0.1 M phosphate buffered saline for 15 min. After dehydration through a graded ethanol series, the slides were stored at -70 °C until used for ISH.
A series of digoxygenin-labelled oligonucleotide probes based on the human gene sequence were used for ISH . These probes, which spanned the two splice junctions in the mRNA, were used to uniquely detect the expression of alternatively spliced mRNA variants in sections from striate cortex of normal and strabismic cats. A 34 base oligomer "pan" probe was designed in antisense orientation from a conserved region of the sequence and was intended to detect all mRNA isoforms. In addition, six other 34 base antisense oligomers were synthesized, based upon the sequences symmetrically spanning the splice junction for each unique isoform. Control probes included sense oligonucleotides and short segment probes that were adjacent to, but not spanning the splice junctions. The probe sequences, GC content and melting temperatures are summarized in Table 1.
The hybridized probe was visualized using a chromogen detection system (Boehringer Mannheim, Germany). A computer assisted systematic morphometric counting procedure was used to enumerate hybridizing cells.
Light microscopic analysis was used (x40 magnification with a field of view 200 x 200 mm). The radial extent of the cortex was covered by three such fields forming a strip. Three such strips were analysed for each slide (chosen at random). Sixty four slides were analysed in toto, 35 from the two strabismic animals (S1, S2) and 29 from the two normal animals (N1, N2). For the strabismic animals, sampling was carried out from hemispheres both ipsilateral (I) and contralateral (C) to the strabismic eye. Thus, in animal S1, 21 slides comprised 11 C and 10 I, while in S2, 14 slides were analysed (9 C, 5 I). Statistical comparison between samples of labelled neurons from strabismic and normal cats was made using analysis of variance (ANOVA).
A total of 66,487 immunopositive cells in the area centralis projection region of the primary visual cortex (Area 17) were counted (with the aid of the computer analysis system) from 64 slides under light microscopy in normal and strabismic cats. ISH for the alternatively spliced isoforms of the NMDAR1 mRNA in the striate cortex of the two normal and two experimental strabismic cats revealed a different distribution of cells stained positively for the heterogeneous variants. There was no signal found by hybridization with the control probe (Figure 1). As expected, the "pan" probe, which detects all the NMDAR1 mRNA species, showed abundant expression in normal visual cortex. The laminar distribution of positive immunostaining for all the hybridized isoforms was most prominent in layer II-III with fewer positively stained cells in the deeper layers IV to VI. This laminar distinction was most noticeable with the isoform R1-3 probe (significant, p<0.05). Intensity of immunoreactive cellular staining across the layers of the normal adult striate cortex was not significantly pairwise different between the probe variants of NMDAR1 mRNA.
Immunopositivity with the "pan" probe of NMDAR1 mRNA was mainly found in the pyramidal and granular neurons of the cortex of normal adult cats (Figure 2). In the strabismic cats, the density of cells showing positive expression of NMDAR1 mRNA was significantly lower than in normals (p<0.0001), an effect observed across all layers, but most notably in layer IV of primary visual cortex. The density of positive NMDAR1 mRNA probes in the visual cortex from strabismic amblyopic cats expressed as a normalized percentage is shown in Figure 3.
The pattern of density of expression of the different isoforms also varied between the cortices of normal and strabismic cats, with decreased proportion of cells staining positively for the R1-a, R1-b, and R1-1 isoforms and increased expression of R1-3 in strabismic cortex (ANOVA, p<0.0001). No significant difference was found in the proportion of cells positively labelled for R1-2 and R1-4 isoforms (p>0.05) in comparing normal and strabismic cortex.
Comparison of the laminar distribution of the mean numbers of neurons (cell/mm2) showed that positive expression of the pan probe of NMDAR1 mRNA, and of isoforms R1-a, R1-b, and R1-1 was markedly less in layers II-III of striate cortex, comparing normal and strabismic cats (Figure 4A). Similarly, the expression of several of the NMDAR1 mRNA isoforms was decreased in layer IV of striate cortex of strabismic cats compared to normal. Notably, cells positively staining for the pan probe, R1-a, R1-b, and R1-1 isoforms were all decreased (Figure 4B). The mean number of neurons positively expressing NMDAR1 mRNA isoforms R1-a, R1-b, and R1-1 in layer V-VI of striate cortex of strabismic cats was reduced compared with normal, while expression of the R1-3 isoform was increased (Figure 4C).
A new variant of the ISH technique using digoxygenin-labelled oligonucleotide probes from human brain sections  has been developed and utilized here to study the changes in gene expression of the NMDAR1 mRNA receptor subtypes of visual cortex of cat. This allows better identification of specifically hybridizing cells with minimal background staining. The alternatively spliced isoforms of NMDAR1 mRNA show heterogeneous expression in the visual cortex of both normal and strabismic cat similar to that which we have previously described for the receptor protein . The localization and distribution of the mRNA isoforms within the layers of visual cortex of normal cat is extremely variable with greatest expression in layers II and III and lesser expression in layers IV through VI.
As expected from our earlier work, we found reduced expression of the pan probe in the cortex of adult strabismic cats compared to normals . This decreased expression was particularly significant for the R1-a, R1-b, and R1-1 isoforms. However, their decrease was accompanied by increased expression of R1-3 and unchanged expression of the R1-2 and R1-4 isoforms. This suggests that the inhibition of transcription of NMDAR1 mRNA in general, and in some specific isoforms in particular, is related to surgical misalignment of the visual axis of one eye early in life. The fact that the greatest difference in immunopositivity between strabismic and normal cortex occurs in layer IV suggests that the change in NMDAR1 receptor proteins probably occurred close to the time of surgery, soon after eye opening, given that layer IV shows greatest plasticity early in the critical period .
While the strabismus operation (by tenotomy) induces amblyopia consistently  and results in much stronger physiologically defined ocular dominance columns than in normals [18,23], the lack of phasic variation in positive NMDAR1 staining tangentially along layer IV shows that both amblyopic and non-amblyopic ocular dominance columns demonstrate lowered staining percentage compared with normal cat, irrespective of whether acuity is normal or not. The most obvious remaining difference between normal and strabismic cortex is the markedly lower percentage of binocular neurons in striate cortex of strabismic cat compared with normals (accompanied by a lowered behavioural stereoacuity). The observation of NMDA antibody column-like staining early in normal development of kitten visual cortex (2 to 5 weeks, vanishing by week 12 ) must be incorporated into this picture. Trepel et al. noted that immunostaining was loosely associated with the boundaries of ocular dominance columns. Such "border" neurons would be most likely to change their properties, including ocular dominance under altered conditions of binocular competition. Thus, the density of NMDA receptors appears to be correlated with the degree of functional plasticity. The relative absence of receptor staining would then suggest less plasticity, confirmed by an early finding that prior strabismus provides protection from the normal (ocular dominance shift) effects of monocular deprivation . Perhaps the necessity for dynamic plasticity in neurons is lessened when stereopsis is absent; neurons in normal cat have to accommodate higher order feedback incorporating object vision in binocular rather than monocular space.
The decreased expression of all isoforms of NMDA mRNA in layer IV of strabismic cat confirms our previously reported finding of decreased numbers of neurons expressing NMDAR1 antibody in all layers of the visual cortex of strabismic cat . This also supports our earlier physiological studies , which suggested that the earliest site in the visual pathway that plastic changes occur in this model of strabismic amblyopia is the main input layer of the visual cortex. The change in transcription of NMDAR1 receptors may contribute to the normal experience dependent modification during development of the neuronal properties of visual cortex. Preliminary findings reported recently on the timing of changes in the NMDA receptor confirm that the number of cells in layer IV expressing the NMDAR1 subunit is reduced compared with normals soon after the misalignment of the visual axes in the kitten . This supports the idea that early strabismus reduces cortical plasticity whereas early dark rearing tends to incubate the potential for plasticity, with dark rearing kittens for 6 weeks resulting in changes in NMDA labelling in cortical layer IV 10 days after bringing the kittens into the light [5,26].
Thus it seems that the distribution of neural NMDA receptors in kitten visual cortex, as well as the binocularity of the neurons themselves , are particularly susceptible to misalignment of the visual axes early in the critical period. It is relevant that NMDAR1-rich patches tended to be associated with the borders of ocular dominance columns , sites where binocularity of neurons is changing most rapidly across cortex. Also, in this strabismus model, physiological response of the amblyopic retinal ganglion cells is normal , but the percentage of binocularly activated neurons is low, due to the lack of synchrony of image information [18,23]. Perhaps the abundance of NMDA receptors maintained in the mature normal cortex is partly due to the dynamic plasticity required for the servicing of such functions as stereopsis, where altered neural output is required depending on whether one or both eyes are contributing input.
The role for NMDA receptors in experience dependent synaptic rearrangement during development is still hypothetical. Nonetheless, it has been shown here that strabismus induced by surgical misalignment of the visual axes in young kittens disrupts the normal pattern of NMDA receptor distribution in the striate cortex of adult cats, particularly in layer IV, where we do not see a differential staining pattern, which should have been obvious if NMDAR1 staining were related to the final ocular dominance of neurons driven by the strabismic and non-deviating eyes. These findings suggest that inhibition of transcription of NMDAR1 mRNA in general and of isoforms R1-a, R1-b, and R1-1 in particular, is the source of the reduction in receptor expression in strabismic cat area 17 with an increase in cells expressing the R1-3 isoform.
This research was supported by an Australian NH& MRC (grant number 970539) to S. G. Crewther and D. P. Crewther and a Chinese NSFC grant to Z. Q. Yin (grant number 39770258). The authors would like to acknowledge with thanks helpful discussion and use of the laboratory facilities of Professor D. Wakefield and Associate Professor A. Lloyd of the Inflammation Research Unit, School of Pathology, University of New South Wales. We would also like to acknowledge the advice of Mr. G. Dixon on preparation of brain sections.
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